PART-[B] Simultaneous reverse phase. high performance liquid. chromatographic [RP-HPLC] determination of salicylamide,

Transcription

1 PART-[B] Simultaneous reverse phase high performance liquid chromatographic [RP-HPLC] determination of salicylamide, salicylic acid and deferasirox in the bulk API dosage forms SECTION-[II] HPLC method development and validation of salicylic acid

2 [1.0] Introduction of salicylic acid (from Latin Salix, willow tree, from the bark of which the substance used to be obtained) is chemically 2-hydroxybenzoic acid. This colorless crystalline organic acid is derived from the metabolism of salicin. Its molecular formula is C 7H 6O 3 and molecular is weight gm/mole. It is poorly soluble in water (2 g/l at 20 C) [1, 2]. It has bacteriostatic, fungicidal, keratolytic actions and widely used in organic synthesis and functions as a plant hormone. The salts and esters of salicylic acid are known as salicylates. Salicylate salt is used as analgesics [3-5]. In addition to being an important active metabolite of aspirin (acetylsalicylic acid), which acts in part as a prodrug to salicylic acid, it is probably best known for its use as a key ingredient in topical anti-acne products. Aspirin (acetylsalicylic acid or ASA) can be prepared by the esterification of the phenolic hydroxyl group of salicylic acid with the acetyl group from acetic anhydride or acetyl chloride. is known for its ability to ease aches and pains and reduce fevers. These medicinal properties, particularly fever relief, have been known since ancient times and it is used as an anti-inflammatory drug [6]. For example, methyl salicylate is used as a liniment to soothe joint and muscle pain and choline salicylate is used topically to relieve the pain of mouth ulcers. Cotton pads soaked in salicylic acid can be used to chemically exfoliate skin as with other hydroxy acids, salicylic acid is a key ingredient in many skin-care products for the treatment of seborrhoeic dermatitis, acne, psoriasis, calluses, corns, keratosis pilaris, acanthosis nigricans, ichthyosis and warts. The standard treatment for all uses is a 6 % aspirin suspension in petroleum jelly, applied on the callus for one hour and then removed with washing. Salicylic acid works as a keratolytic, comedolytic and bacteriostatic agent, causing the cells of the epidermis to shed more readily, opening clogged pores and neutralizing bacteria within, preventing pores from clogging up again by constricting pore diameter and allowing room for new cell growth [7, 8]. Use of concentrated solutions of salicylic acid may cause hyper pigmentation on unpretreated skin for those with darker skin types, as well as with the lack of use of a broad spectrum sun block [9, 10]. Bismuth subsalicylate, a salt of bismuth and salicylic acid, is the active ingredient in stomach relief aids such as Pepto-Bismol, is the main ingredient of Kaopectate and displays anti- PART-[B] Page 72

3 inflammatory action (due to salicylic acid) and also acts as an antacid and mild antibiotic [11-13]. It is derived from a bitter powder that comes from the bark of a willow tree. Though it does not have any effect on the acne bacteria in the pores or the excess production of sebum that cause acne, 1.5 % - 2 % concentrations of salicylic acid are effective at clearing mild to moderate acne effectively without over drying the skin. In some skin creams for dry or mature skin, salicylic acid is used in lower concentrations as an exfoliating ingredient which increases the efficacy of active ingredients. Since salicylic acid can be slightly drying to the surface of the skin, it is advised not to use it along with topical acne prescriptions, aggressive cleansers and astringents. For the best results, a balanced acne regimen with moisturizing and soothing products is recommended along with salicylic acid products. The P.acnes bacterium that causes acne cannot live in an oxygen rich environment which benzoyl peroxide when it comes in contact with the skin so breakouts are reduced. It is also a keratolytic, providing exfoliating benefits in the pores to prevent clogging. It is used in 2.5 %, 5 % and 10 % concentrations depending on the severity of acne and sensitivity level of the skin although higher concentrations are not always more effective. Benzoyl peroxide can be found in many over the counter creams, gels and lotions as well as in stronger prescription products. It can also bleach fabric so it s best to protect your clothing and linens when using products with this ingredient. Side effects range from mild to severe dryness, peeling and swelling so careful selection of other skin care products should be made so as not to increase the severity of these effects. [2.0] Chemical structure, IUPAC and common name of salicylic acid PART-[B] Page 73

4 Common name 2-Carboxyphenol, 2-hydroxy-benzoic acid, o-carboxyphenol, o-hydroxybenzoic acid, salicylic acid, SAX, Ionil, Keralyt, Saligel, Salonil, Duoplant, Freezone, Rutranex, Stri-Dex and verrugon [3.0] Brief overview of synthetic pathway of salicylic acid is biosynthesized from the amino acid phenylalanine. In arabidopsis thaliana it can also be synthesized via a phenylalanine-independent pathway. Sodium salicylate is commercially prepared by treating sodium phenolate with carbon dioxide at high pressure (100 atm) and high temperature (125ºC), a method known as the Kolbe-Schmitt reaction. Acidification of the product with sulfuric acid gives salicylic acid. It can also be prepared by the hydrolysis of aspirin (acetylsalicylic acid or methyl salicylate (oil of wintergreen) with a strong acid or base. [4.0] Description [4.1] Pharmacodynamics treats acne by causing skin cells to slough off more readily, preventing pores from clogging up. This effect on skin cells also makes salicylic acid an active ingredient in several shampoos meant to treat dandruff. Subsalicylates in combination with bismuth form the popular stomach relief aid known commonly as Pepto-Bismol. When combined the two key ingredients help control diarrhea, nausea, heartburn, and even gas. It is also very mildly anti-biotic. [4.2] Mechanism of action directly and irreversibly inhibits the activity of both types of cyclo-oxygenases (COX-1 and COX-2) to decrease the formation of precursors of prostaglandins and thromboxanes from arachidonic acid. is a key ingredient in many skin-care products for the treatment of acne, psoriasis, PART-[B] Page 74

5 calluses, corns, keratosis pilaris, and warts. Because of its effect on skin cells, salicylic acid is used in several shampoos used to treat dandruff. shows its anti-inflammatory effects via suppressing the activity of cyclooxygenase (COX), an enzyme that is responsible for the production of pro-inflammatory mediators such as the prostaglandins. It does this not by direct inhibition of COX like most other non-steroidal antiinflammatory drugs (NSAIDs) but instead by suppression of the expression of the enzyme [11]. has also been shown to activate adenosine monophosphate-activated protein kinase (AMPK), and it is thought that this action may play a role in the anticancer effects of the compound and its prodrugs aspirin and salsalate. In addition, the antidiabetic effects of salicylic acid are likely mediated by AMPK activation primarily through allosteric conformational change that increases levels of phosphorylation [12]. Salicylic acid also uncouples oxidative phosphorylation, which leads to increased ADP : ATP and AMP : ATP ratios in the cell. As a consequence, salicylic acid may alter AMPK activity and subsequently exert its anti-diabetic properties through altered energy status of the cell. Even in AMPK knock-out mice, however, there is an anti-diabetic effect, demonstrating that there is at least one additional, yet-unidentified action of the compound [13]. Table 1: Physical and chemical properties of salicylic acid Physical and chemical properties Property Value Molecular Weight g/mol Molecular Formula C 7H 6O 3 Density 1.44 g/cm 3 Solubility Easily soluble in methanol CAS number Physical state colorless to white crystals Melting point C Boiling point 211 C Stability Stable, substances to be avoided include oxidizing agents, strong bases, iodine, fluorine, combustible, Sensitive to light. Categories Antifungal agents, anti-infective agents, keratolytic agents PART-[B] Page 75

6 Prescription, indications and usage of salicylic acid 6 % is a topical aid in the removal of excessive keratin in hyperkeratotic skin disorders including verrucae, and the various ichthyoses (vulgaris, sex-linked and lamellar), keratosis palmaris and plantaris keratosis pilaris, pityriasis rubra pilaris, and psoriasis. gel is used to treat scalp itching, flaking, irritation, redness, and scaling due to psoriasis, dandruff, or seborrhea. It may also be used for other conditions as determined by your doctor. gel is a topical salicylate. Topical salicylic acid comes as a cloth, cream, lotion, liquid, gel, ointment, shampoo, wipe, pad, and patch, several strengths, including certain products that are only available with a prescription to apply to the skin or scalp. It treats other skin conditions by softening and loosening dry, scaly, or thickened skin so that it falls off or can be removed easily. It is used to help clear and prevent pimples and skin blemishes in people who have acne. It is also used to treat skin conditions that involve scaling or overgrowth of skin cells such as psoriasis, ichthyoses, dandruff, corns, calluses, and warts on the hands or feet. is in a class of medications called keratolytic agents. It may be used as often as several times a day or as infrequently as several times a week, depending on the condition being treated and the products are being used. Table 2: topical dosage Topical Dosage topical 1 % pad topical 16.7 % liquid 3 % topical soap topical 6 % cream topical 6 % lotion topical 6 % foam Cleanse affected area; apply 2 to 3 times daily. If dryness occurs, reduce to every other or once a day Wash and dry area thoroughly. Apply enough to cover each wart 1 to 2 times daily Apply to affect areas at least twice weekly. Leave lather on scalp or skin for two minutes then rinse Apply to affected area once daily. Hydrate area for 5 minutes prior to application if possible. Occlude the area at night. Wash off in morning Apply to affected area once daily. Hydrate area for 5 minutes prior to application if possible. Occlude the area at night. Wash off in morning Apply to affected area once daily at bedtime. Hydrate area for 5 minutes prior to application if possible. Occlude the area after application. Wash off in morning PART-[B] Page 76

7 - clinical pharmacology Salicylic Acid has been shown to produce desquamation of the horny layer of skin while not effecting qualitative or quantitative changes in the structure of the viable epidermis. The mechanism of action has been attributed to dissolution of intercellular cement substance. Follow the directions on the package label or your prescription label carefully, and ask your doctor or pharmacist to explain any part you do not understand. Use salicylic acid exactly as directed. Do not use more or less of it or use it more often than directed on the package or prescribed by your doctor. If you are using topical salicylic acid to treat acne, your skin may become dry or irritated at the beginning of your treatment. To prevent this, you may apply the product less often at first, and then gradually begin to apply the product more often after your skin has adjusted to the medication. If your skin becomes dry or irritated at any time during your treatment, you may apply the product less often. [5.0] Survey of analytical method/literature reviews The literature reviews regarding salicylic acid suggest that various analytical methods were reported for its determination in pharmaceutical formulation and in various biological fluids. As per discussion in the literature reviews UV, LC- MS, HPLC methods for the determination of salicylic acid in pharmaceutical dosage forms are reported. The analytical methods including UV, HPLC, HPTLC, LC-MS/MS, GC and fluorimetric have been reported for the estimation of salicylic acid alone or simultaneously with other drugs. However no method has been reported for simultaneous estimation of salicylamide, salicylic acid and deferasirox. The present work describes simultaneous estimation of salicylamide, salicylic acid in bulk API pharmaceutical dosage forms [14-31]. The literature reviews for analysis of salicylic acid are as under: [1] PH. Patel, B. Shrivastava, J. Prajapati, J. Akhtar et al, have developed and validated a simple, precise and accurate Rp-HPLC method for simultaneous estimation of niacinamide and salicylic acid in semi solid dosage form. The separation is done by using column oyster BDS C-18 (4.6 x 250 mm, 5 µm, 110 Å) at 25ºC and 74 : 26 (V/V) water: methanol as mobile phase at flow rate of 1 ml/min and ph adjusted with triethylamine and glacial acetic acid. Detection is carried out at 280 nm. The method has been validated according to ICH PART-[B] Page 77

8 guideline in terms of linearity, precision, accuracy, specificity and solution stability. The linearity of proposed method is investigated in the range of µg/ml (R 2 = %) for niacinamide and µg/ml (R 2 = %) for salicylic acid. The percentage recoveries of niacinamide and salicylic acid are found to be % and % respectively. The proposed method provides an accurate and precise quality control tool for analysis of niacinamide and salicylic acid in semisolid dosage forms [32]. [2] JS. Mary, Vimal Kumar et al, have developed and validated a rapid reversed-phase high-performance liquid chromatographic procedure for the simultaneous quantitation of aspirin, salicylic acid, and caffeine extracted from an effervescent tablet. The method uses a Hypersil C18 column (5 μm, 15 cm 4.6 mm) for an isocratic elution in a water : methanol : acetic acid mobile phase at a wavelength of 275 nm. The tablets buffering effects and acid neutralizing capacity require an extraction solvent of methanol : formic acid. The range of linearity for aspirin is mg/ml, caffeine mg/ml, and salicylic acid % of aspirin. The overall recovery is %, %, and 99.2 % for aspirin, caffeine, and salicylic acid, respectively. Under the conditions of the method, aspirin, caffeine, and salicylic acid are adequately resolved with proper peak symmetry in less than 7 min. [33]. [3] B. Patel, H. Raj, V. Jain, V. Sutariya, M. Bhatt et al, have developed and validated a simple, gradient, rapid and accurate reversed phase high performance liquid chromatography method for simultaneous determination of Clobetasol propionate and salicylic acid in its pharmaceutical dosage forms. Unisphere C-18 column (5 μm, 250 mm x 4.6 mm i.d.) at 45 ºC temperature and UV detector at 240 nm was used. (Methanol: Sodium dihydrogen phosphate monohydrate (ph 5.5 adjusted with NaOH): acetonitrile (6 : 29 : 65) was used as mobile phase with 1 ml/min with injection volume 20 μl. The method has been validated according to ICH guidelines. Calibration graph was found to be linear in μg/ml and 5-15 μg/ml for salicylic acid and clobetasol propionate respectively. The regression coefficient (R 2 ) obtained was found to be % for both drugs. The retention time were found to be min and min respectively [34]. [4] Safeena Sheikh, Suhail Asghar, Showkat Ahmad Patni et al, have developed and validated a stability indicating high performance liquid PART-[B] Page 78

9 chromatography (HPLC) method for the quantification of salicylic acid (SA) and tolnaftate (TF) in combined pharmaceutical ointment base formulations. The separation was performed on a Merck C-18 column with the mobile phase consisting of Acetonitrile : Methanol : Water (50 : 20 : 30 v/v) at flow rate 1.5 ml/min. Both the drugs were resolved successfully with retention time min and min, when detection was carried out at UV 245 nm. The overall retention times of analytes were 10.0 minutes. The method was validated with respect to linearity, precision, accuracy and recovery. The relative standard deviation for six replicate measurements of SA and TF were % and % respectively. Total recoveries of analytes were %, %, % and %, %, % of SA and TF respectively when examine over the range of 80 %, 100 %, and 120 % of added drugs in placebo. The linearity of SA and TF were found in the range of μg/ml and 32.0 μg/ml to 48.0 μg/ml respectively [35]. [5] M. Shou, WA. Galinada, Yu-ChienWei, Q. Tang, RJ. Markovich, AM. Rustum et al, developed a reversed-phase high performance liquid chromatography (RP-HPLC) method for simultaneous determination of salicylic acid, betamethasone dipropionate, and their related compounds in diprosalic lotion. A 150 mm 4.6 mm I.D. YMC J sphere ODS-H80 column at 35 C and UV detection at 240 nm was used. A gradient elution was employed using 0.05 % (v/v) methanesulfonic acid solution and acetonitrile as mobile phases. A total of thirty three compounds from diprosalic lotion samples were separated in 38 min. [36]. [6] Pushpa Kumari K, Gowri Sankar, P. Sowjanya, S. Madhubabu et al, have developed and validated a stability indicating RP-HPLC method for the determination of salicylic acid in trilisate tablets using Xorabax XBD C18 column ( mm, 5 μm) with mobile phase consisting of phosphate buffer (ph 3.0) : methanol (80 : 20 v/v) with a flow rate of 1.0 ml/min. Detection was carried out at 230 nm. Retention time was found to be 4.6 min. Linearity was observed over the concentration range of 5-30 μg/ml (R 2 = %) with regression equation y = 30.55x was subjected to stress conditions including acidic, alkaline, photolysis and thermal degradation. The drug is more sensitive towards alkaline degradation. The method was validated as per ICH guidelines [37]. PART-[B] Page 79

10 [7] FH. Havaldar, DL. Vairal et al, have developed and validated a simple, specific, accurate and economical isocratic reversed phase liquid chromatographic (RP-HPLC) method for the determination of paracetamol, acetyl salicylic acid, mefenamic acid and cetirizine dihydrochloride. Separation was achieved with a Nucleodur 100 C 18 column having 250 x 4.6 mm i.d. with 5 μm particle size and disodium hydrogen phosphate buffer adjusted to ph 6.5 using diluted orthophosphoric acid and acetonitrile (60 : 40 v/v) as eluent at a constant flow rate of 1.0 ml per min. UV detection was performed at 220 nm. The retention time of acetyl salicylic acid, paracetamol, mefenamic acid and cetirizine dihydrochloride were 2.01 min, 2.92 min, 4.91 min and 10.2 min respectively. This method is simple, rapid and selective and can be used for routine analysis of analgesic and antipyretic drugs in pharmaceutical formulations [38]. [6.0] Aim and scope of present work The primary objective of the present work was thus to develop and validate a RP-HPLC method for the assay of salicylic acid from API dosage forms. Hence, the method is useful for routine quality control analysis and also for determination of stability. Purpose of the present study was to develop and validate a RP-HPLC method for determination of salicylic acid in API pharmaceutical dosage forms. The aim and scopes of the proposed work are as under: 1. To select suitable mobile phase (solvent buffer ratio) 2. To optimize RP-HPLC conditions 3. To develop suitable HPLC method for salicylamide 4. Perform the validation for the developed method [7.0] Experimental [7.1] Materials & Orthophosphoric acid were obtained from s d Fine Chemical Limited. Acetonitrile (fisher Qualigens, HPLC grade) were obtained from Thermo Fisher Scientific India Pvt. Ltd. and potassium dihydrogen phosphate was obtained from (Merck Specialties Private Limited). PART-[B] Page 80

11 [7.2] Equipment Equipment Apparatus HPLC System Dionex ultimate 3000 (Germany) High performance liquid chromatographic system equipped with ultimate 3000 Pump, Auto Sampler, Column Compartment and RS Diode Array Detector Software Dionex Chromeleon 7 (Version 7.1, Simply Intelligent) Column oven Ambient Column Waters symmetry C18 (4.6 x 250mm, 5µm, 110 Å) [7.3] Preparation of stock and sample solutions [7.3.1] Preparation of buffer Potassium dihydrogen phosphate (1.36 g) was dissolved in 1000 ml high purity demonized Milli-Q water [Millipore, Milli-Q, Bedford, MA, USA, purification system] and ph was adjusted 3.2 with ortho-phosphoric acid and filtered through 0.22 μ size nylon filter under vacuum. [7.3.2] Preparation of mobile phase The mobile phase was prepared by mixing 400 ml phosphate buffer ph 3.2 and 600 ml of acetonitrile [HPLC Grade]. The mixture was sonicated in Expo-Hi Tech sonicator for 5 minutes. [7.3.4] Preparation of standard and sample solution The Standard stock solutions were prepared by accurately weighing 100mg of each salicylic acid in 100 ml volumetric flask (1000 µg/ml) in acetonitrile. Sample solutions were prepared by appropriate dilution of the standard solutions with the diluent. [8] Method development and optimization of chromatographic conditions (UV graph/chromatograms) To develop a precise, accurate and suitable RP-HPLC method for the estimation of salicylic acid different mobile phases, solvent-buffer ratios and ph were tried to proposed final chromatographic conditions. Deferasirox is soluble in methanol and acetonitrile-water mixture. The peak shape, resolution and symmetry of salicylic acid were good with above gradient elution at a 1.0 ml/min flow rate. The method developed was unique in determining the impurities even at low levels than that of specifications. The developed method was successfully applied to estimate the amount of deferasirox. PART-[B] Page 81

12 Optimized chromatographic conditions Parameter Optimized condition Flow rate 1.0 ml/min Mobile phase 40 : 60 v/v (Buffer : ACN) Buffer ph Potassium phosphate buffer ph 3.2 adjusted by OPA Wavelength 245 nm Injection volume 10 µl Run time 15 min Column and column 30ºC oven temperature Figure 1: Chromatogram and UV calibration curve for salicylic acid standard (Mobile phase: ACN : buffer, 60 : 40 v/v, ph 3.2 with OPA) PART-[B] Page 82

13 Figure 2: Chromatogram and UV calibration curve for salicylic acid sample (Mobile phase: ACN : buffer, 60 : 40 v/v, ph 3.2 with OPA) Figure 3: Chromatogram and UV calibration curve for salicylic acid sample (Mobile phase: ACN : buffer, 60 : 40 v/v, ph 3.2 with OPA). PART-[B] Page 83

14 Figure 4: Chromatogram and UV calibration curve for salicylic acid sample (Mobile phase: CAN : buffer, 60 : 40 v/v, ph 3.2 with OPA). PART-[B] Page 84

15 [9] Analytical method validation The optimized RP-HPLC assay method was validated for specificity, linearity, accuracy, precision (repeatability and intermediate precision), recovery and system suitability according to International Conference on Harmonization (ICH) guidelines for the validation of bioanalytical method [39] and the US Food and Drug Administration (FDA) [40]. [9.1] System suitability System suitability was performed by using 100 µg/ml of salicylic acid by making six replicate injections. Chromatographic parameters calculated from experimental data, such as Number of theoretical plates, % RSD of peak area and resolution factors (Rs) are given in table-3. The system was deemed to be suitable for use if the capacity factors were in the range of (11.77 <K < 20), lower than 2 for tailing factor, more than 2 for resolution (Rs), greater than 4357 number of theoretical plates (N), resolution between salicylic acid of at least two and less than 2 % relative standard deviation (% RSD) for peak area. Table 3: System suitability parameters System suitability parameters Sr. No. Parameters 1 Linearity range (µg/ml) µg/ml 2 Retention time (Min.) Theoretical plates (N) Peak Asymmetry (T) Resolution (Rs) Accuracy % 7 Precision % 8 % RSD (For peak area) % [9.2] Precision The precision of an analytical method is the degree of agreement among individual test results when the method is applied repeatedly to multiple samplings of homogenous samples. This is usually expressed as the standard deviation or the relative standard deviation. The precision of the assay was studied with respect to both intra-day (Repeatability) and Inter-day (Intermediated) precisions. Repeatability was calculated from five replicate injections of three different concentrations of salicylic acid in the same equipment on the same day. Inter day precision was PART-[B] Page 85

16 checked with the same concentrations as intra-day assay and the determination of each compound was repeated day by day during three days. The method was found to be precise with RSD values within for intra-day and inter day assay. Evaluation of the intra-day and inter-day precision for the determination of salicylic acid by the proposed HPLC method according to ICH guidelines. Intra Day (Repeatability) precision Repeatability can be defined as the precision of the procedure when repeated by same analyst under the same operating conditions over a short interval of time or same day. It is normally expected that at least six replicates be carried out and individual result provided from mean, standard deviation and coefficient of variation should be calculated for set of n value. The RSD values are important for showing degree of variation expected when the analytical procedure is repeated several time in a standard situation (RSD below 2 % for assays in finished product). Inter day (Intermediate) precision Repeatability can be defined as the precision of the procedure when repeated by same analyst under the same operating conditions and the determination of each compound was repeated day by day during three days or study repeat three days over a long interval of time. Table 4: Intra-day and Inter-day precision data for salicylic acid sample Sample Con. (µg/ml) Intra day Area (mau*m in) Mean area % SD % RSD Inter day Area (mau*m in) Mean area % SD % RSD PART-[B] Page 86

17 Table 5: Intra-day and Inter-day precision data for salicylic acid standard Standard con. Intra day Inter day (µg/ml) Area (mau*min) Area (mau*min) Average % SD % RSD Standard potency % [1] % Assay = P AT = Average area of obtained in sample preparation AS = Average area of obtained in standard preparation W1 = Weight taken of reference standard (mg) W2 = Weight taken of test sample (mg) P = Potency of reference standard (%) [2] [1] Intra day 1. % Assay = = = % 2. = = % [2] Inter day 1. % Assay = = = % 2. = = % [9.3] Limit of detection (LOD) and Limit of Quantification (LOQ) The limit of detection (LOD) is defined as the lowest concentration of an analyte that can reliably be differentiated from background levels. The standard solutions of the compounds for LOD were prepared by diluting them sequentially. Limit of quantification (LOQ) of an individual analytical procedure is the lowest amount of analyte that can be quantitatively determined with PART-[B] Page 87

18 suitable precision and accuracy (ICH Guideline Q2B, 2005). LOD and LOQ were determined calculating the signal-to-noise ratio of each compound by injecting a series of solution until the S/N ratio 3 for LOD and 10 for LOQ. where S is the standard deviation of y-intercepts of regression. [9.4] Specificity Specificity of method can be absence of any interference at retention times of samples. The specificity of the method was demonstrated by injection of standard solution of salicylic acid at concentration of 100 µg/ml. The elution peaks of salicylic acid presented in representative chromatograms shown in Figure 4. The representative chromatogram for simultaneous determination of the studied drugs in API pharmaceutical dosages forms. Table 6: Specificity study of salicylamide Con. (µg/ml) Sample Standard Area (mau*min) Area (mau*min) Average % SD % RSD Standard potency % [1] % Assay = = = % [2] = = % [9.5] Linearity The linearity of salicylic acid was studied by preparing standard solution at five different concentrations ranging from µg/ml. Each concentration was injected in a five replicates and mean value of peak area was taken for calibration curve. Construction of the calibration curves Working solutions containing ( ) μg/ml were prepared by serial dilution of standard solution with the acetonitrile. In all cases, 10 µl PART-[B] Page 88

19 aliquots were injected (triplicate) and eluted with the mobile phase under the following chromatographic conditions. The average peak area ratio of each drug and the internal standard were plotted versus the final concentration of the drug in μg/ml to get the calibration graph. Alternatively, the corresponding regression equation was derived. Sr. No. Concentration (µg/ml) Area (mau*min) Figure 5: Linearity curve PART-[B] Page 89

20 Table 7: Summary of linearity data Summary of linearity data Sr. No. Parameters 1 Linearity range (µg/ml) µg/ml 2 Slope ± Standard error Intercept ± Standard error x 4 Linearity equation Y = x R [9.6] Accuracy Accuracy of the assay method was calculated for salicylic acid by recovery studies at three concentrations of 50 %, 100 %, 150 % and 200 % levels by standard addition method. The mean % recovery for salicylamide, salicylic acid and deferasirox were found is given in the following table. Table 8: Sample and standard area for salicylic acid Sample Area (mau*min) Standard Area (mau*min) 50% 100% 150% 200% 1000% Average % SD % RSD Standard potency % [1] Amount added (µg/ml) = = = µg/ml [2] Amount found (µg/ml) = = = µg/ml [3] % Recovery = = % PART-[B] Page 90

21 Table 9: Accuracy data for salicylic acid Sr. Salicylic Added Found % % Mean SD % RSD No. acid (µg/ml) (µg/ml) Recovery Recovery 1 50% % % , % [10.0] Summary and Conclusion An accurate, sensitive, and precise RP-HPLC method with PDA detection was developed and validated for salicylic acid in their bulk API dosages forms. The proposed method is very rapid, where the total analytical run time for salicylic acid (R t = min.) and the internal standard is less than 10 min. The method can also be readily adapted to routine quality control analysis. [11.0] References [1] inchem.org [2]. Drugbank.ca [3] The Indian pharmacopeia, New Delhi, The Controller of publication. 2010, [4] E. Mikami, T. Goto, T. Ohno, H. Matsumoto, M. Nishida. J. Pharm. Biomed. Anal. 2002, 28, [5] N.C. Desai, R.D. Senta. Simultaneous Rp-HPLC determination of salicylamide, salicylic acid and deferasirox in the bulk API dosages forms. Journal of Taibah University for Science.9, 2015, PART-[B] Page 91

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